Nonhepatic origin of notothenioid antifreeze reveals
pancreatic synthesis as common mechanism in polar
fish freezing avoidance
Chi-Hing C. Cheng*†, Paul A. Cziko*, and Clive W. Evans‡
*Department of Animal Biology, University of Illinois, Urbana, IL 61801; and‡Molecular Genetics and Development, School of Biological Sciences,
University of Auckland, Auckland, New Zealand
Communicated by George N. Somero, Stanford University, Pacific Grove, CA, May 9, 2006 (received for review March 30, 2006)
Phylogenetically diverse polar and subpolar marine teleost fishes
have evolved antifreeze proteins (AFPs) or antifreeze glycopro-
teins (AFGPs) to avoid inoculative freezing by internalized ice. For
over three decades since the first fish antifreeze (AF) protein was
discovered, many studies of teleost freezing avoidance showed
hepatic AF synthesis and distribution within the circulation as
pivotal in preventing the blood, and therefore the fish, from
freezing. We have uncovered an important twist to this long-held
paradigm: the complete absence of liver synthesis of AFGPs in any
life stage of the Antarctic notothenioids, indicating that the liver
plays no role in the freezing avoidance in these fishes. Instead, we
found the exocrine pancreas to be the major site of AFGP synthesis
and secretion in all life stages, and that pancreatic AFGPs enter the
intestinal lumen via the pancreatic duct to prevent ingested ice
from nucleating the hyposmotic intestinal fluids. AFGPs appear to
remain undegraded in the intestinal milieu, and the composition
and relative abundance of intestinal AFGP isoforms are nearly
identical to serum AFGPs. Thus, the reabsorption of intact pancre-
as-derived intestinal AFGPs, and not the liver, is the likely source of
circulatory AFGPs in notothenioid fishes. We examined diverse
northern fish taxa and Antarctic eelpouts with hepatic synthesis of
bloodborne AF and found that they also express secreted pancre-
atic AF of their respective types. The evolutionary convergence of
this functional physiology underscores the hitherto largely unrec-
ognized importance of intestinal freezing prevention in polar
teleost freezing avoidance, especially in the chronically icy Antarc-
antifreeze glycoprotein-null liver ? antifreeze paradigm shift ? evolutionary
adaptation ? intestinal freeze avoidance ? functional convergence
(20–35 mg?ml) of protein antifreeze (AF) (a family of AF glyco-
proteins; AFGPs) in their blood and other body fluids (4, 5). The
synthesis of blood AFGPs in Antarctic notothenioids has histori-
cally been attributed to the liver (6, 7), because the vertebrate liver
is well known as the major source of secreted plasma proteins (8,
9), and thus there were no a priori reasons to invoke a different
source for the abundant plasma AFGPs. Also contributing to the
prevailing notion of universal hepatic AF synthesis is the readily
demonstrable liver expression of AF mRNA by Northern blots
and?or cDNA cloning in all other AF protein (AFP)-bearing fish
AFGP-bearing polar cod (15). However, definitive verification of
liver biosynthesis of AFGPs in Antarctic notothenioids has been
lacking. Early radioactive-tracer investigations of notothenioid
AFGP biosynthesis could not rule out a nonhepatic synthesis site,
because the appearance of labeled AFGPs and non-AFGP plasma
proteins in the blood was drastically asynchronous (AFGPs lagged
nototheniid Notothenia coriiceps showed AFGP mRNA hybridiza-
tion, but an unusually large amount (50 ?g) of polyA? RNA was
he predominant endemic Antarctic marine teleost group, the
notothenioid fishes (1), inhabit the world’s coldest and iciest
required (16), contradictory to liver being a strong expression site.
If there is little or no hepatic AFGP biosynthesis, the tissue origin
of the abundant bloodborne AFGPs in Antarctic notothenioids
returns as an unsolved fundamental question ?30 years after the
discovery of the protein (5). In our prior elucidations of the
evolution of the notothenioid AFGP gene from a pancreatic
TLP cDNAs were obtained from exocrine pancreas RNA, indicat-
ing that exocrine pancreas is an AFGP expression site (17, 18). In
this comprehensive study, we confirm that the exocrine pancreas is
the major AFGP synthesis site in Antarctic notothenioid fishes
from hatching through adulthood, whereas the liver is AFGP-
expression null in all life stages. We show that pancreatic AFGPs
are secreted into the intestine, and, with additional AFGP contri-
bution from the anterior portion of the stomach (the only other
icy Antarctic waters. The apparently undegraded intestinal AFGPs
raise the possibility that plasma AFGPs in the notothenioids are
derived from reabsorption of these intact macromolecules, which
despite the absence of liver AF synthesis and secretion. In addition,
we examine diverse AF-bearing species from both north- and
south-polar and subpolar regions and confirm that they have
which attests to intestinal freezing prevention as a vital and integral
component of the repertoire of teleost freeze-avoidance strategies.
AFGP mRNA Expression in Adult Notothenioid Tissues. To determine
the tissue site or sites of AFGP synthesis in adult notothenioids,
Northern blots of total RNA from different tissues were hybridized
to an AFGP gene (coding region only) probe (Fig. 1). Like the
phyletically basal AFGP-null New Zealand notothenioid Bovichtus
each of the five endemic Antarctic notothenioid families (Noto-
theniidae, Artedidraconidae, Harpagiferidae, Bathydraconidae,
and Channichthyidae; detailed species information in Table 1,
showed no hybridization, as opposed to the intense hybridization in
pancreatic RNA sampled from the Antarctic nototheniid Dissos-
tichus mawsoni included in the blot (Fig. 1A Upper). As a control,
Conflict of interest statement: No conflicts declared.
Abbreviations: AF, antifreeze; AFGP, antifreeze glycoprotein; AFP, antifreeze protein; GI,
gastrointestinal; IF, intestinal fluid; PF, pancreatic fluid; TLP, trypsinogen-like protease.
Data deposition: The 26 sequences reported in this paper have been deposited in the
GenBank database (accession nos. DQ062435–DQ062459 and DQ394083). Details listed in
Table 4, which is published as supporting information on the PNAS web site.
†To whom correspondence should be addressed at: Department of Animal Biology,
515 Morrill Hall, 505 South Goodwin Avenue, University of Illinois, Urbana, IL 61801.
© 2006 by The National Academy of Sciences of the USA
July 5, 2006 ?
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(Fig. 1A Lower).
In contrast to liver RNA, Northern blot of RNA from pancreatic
tissues of species representing the five endemic Antarctic notothe-
nioid families showed strong AFGP mRNA expression (Fig. 1B
Upper). Because adult teleost exocrine pancreas is commonly
diffuse and scattered beneath the surface or between layers of
mesenteric (peritoneal or serosal) and fatty tissues associated with
abdominal organs and the biliary and pancreatic ducts (19, 20), we
confirmed the presence of pancreatic mRNA in our pancreatic
tissue isolates based on the positive hybridization with a probe
derived from a D. mawsoni pancreatic trypsinogen cDNA (Fig. 1B
Lower). Lower gastrointestinal (GI) tract components (pyloric ceca
and intestine) with surface mesentery removed showed no AFGP
expression (Fig. 1B Upper). The sizes of pancreatic AFGP mRNAs
ranged from ?1.35 kb to ?9.5 kb (Fig. 1B Upper), reflecting the
chimeric AFGP-TLP genes found in extant Antarctic notothenioid
Antarctic bathydraconid Gymnodraco acuticeps identified the an-
terior portion of the stomach immediately next to the esophagus-
stomach junction as the only other strong expression site of AFGP
mRNA in adults (Fig. 1C).
AFGP Expression in Larval Notothenioid Fish. Immunodetection of
mature AFGPs using polyclonal rabbit anti-AFGP antibodies and
fluorescently labeled secondary goat anti-rabbit IgG in tissue
sections of a 1-day-old G. acuticeps hatchling (Fig. 2 A–D and G)
and a 4-month-old larva (Fig. 2 E, F, and H) showed no immuno-
reactive AFGPs in the liver cells of either age, but both ages
revealed a high degree of AFGP expression in pancreatic acinar
cells, particularly within apical zymogen granules. Pancreatic
AFGP-positive and hepatic AFGP-null expression is therefore not
an adult-only condition, but occurs at hatching and in the early life
stages of notothenioid fishes.
Osmolarity and AF Activity of GI Fluids. The strong AFGP mRNA
expression in the adult exocrine pancreas and anterior stomach
(Fig. 1) is associated with the presence of significant AF activity
(measured as thermal hysteresis, the difference between melting
2, which is published as supporting information on the PNAS web
site), indicating that transcripts are translated into secreted pro-
teins. The stomach fluid is isosmotic or moderately hyposmotic
the five Antarctic notothenioid families (lanes 2–5) and the basal AFGP-null New Zealand species Bv (B. variegatus) (lane 6), versus strong pancreatic expression
liver samples (Lower). (B) AFGP expression in pancreatic tissue isolates from selected species from all five Antarctic notothenioid families and no expression in
nonpancreatic GI components (Upper). Location of pancreatic tissue isolates: st, stomach; pc, pyloric ceca; gb, gall bladder; gi, GI locations; ant, anterior; post,
posterior. Hybridization of pancreatic RNA samples to a pancreatic trypsinogen probe verifies presence of exocrine pancreas RNA (Lower). (C) AFGP expression
in anterior stomach (lanes 1 and 2) and absence of expression in all other tissues from Ga (G. acuticeps). (Detailed species information is in Table 1.)
Northern blot analysis of AFGP mRNA expression in adult notothenioid fish tissues. (A) Absence of liver AFGP expression in five species representing
acuticeps larvae. (A–D) One-day-old larva. (A) Phase-contrast image showing
DAPI-stained image illustrating the position of nuclei (blue). (C) Immunola-
DAPI, and immunofluorescence). The inset (3? magnification) shows AFGP
localization in some (open arrow) but not all zymogen granules (filled arrow)
of the liver. Immunostained AFGP is lacking. (F) Composite image of the
granules of acinar cells (open arrow). (G) Image of a 1-day-old hatchling. (H)
Image of a 4-month-old larva.
Immunodetection of AFGP expression in Antarctic notothenioid G.
www.pnas.org?cgi?doi?10.1073?pnas.0603796103 Cheng et al.
(1,039?21 to 772?151 mOsm) to seawater (1,025 mOsm), reflect-
dilution by the incipient digestion in the stomach. Intestinal fluid
(IF) is significantly hyposmotic (603?65 to 748?27 mOsm) to
seawater, reflecting dilution by GI secretions, late-stage digestion,
and intestinal salt absorption (21). The presence of AFGPs in the
stomach fluid and IF depresses their freezing points to below that
neither fluid risks inoculative freezing by ingested ice. Thermal
indicating the presence of abundant AF. Pancreatic fluid (PF) was
only obtained from the large nototheniid D. mawsoni in which the
pancreatic duct (distinct from the bile duct) visibly distends into a
small reservoir at its base above its junction with the intestine. D.
mawsoni PF displays a large thermal hystersis (0.94?0.38°C),
indicative of the presence of substantial AFGPs before secretion
into the intestinal lumen.
of size isoforms (15, 22), and the profiles of electrophoretically
resolved AFGPs purified from the PF and IF of one D. mawsoni
specimen show they comprise the full complement of isoforms
similar to serum AFGPs of the same specimen in terms of size
IF AFGP profiles support the pancreas as a major source of
intestinal AFGPs, with the input of stomach AFGPs to the IF
contributing to the observed IF profile (Fig. 4A). Intestinal AFGP
from the anterior and posterior half of the intestine respectively
(Fig. 4A). Profiles of IF and serum AFGPs are nearly identical,
suggesting the possibility that the latter may be derived from the
former via reabsorption. For species of the other four Antarctic
notothenioid families, which are too small for PF sampling, we
cloned and sequenced partial pancreatic AFGP cDNAs from a
representative member of each family. All pancreatic cDNAs
encode AFGPs that share a conserved signal peptide sequence
(Fig. 6, which is published as supporting information on the PNAS
web site), indicating their translation into secreted proteins. Pan-
creatic AFGP expression and secretion therefore occurs through-
out members of all the Antarctic notothenioid families.
Pancreatic AF Expression in Nonnotothenioid AF-Bearing Fishes. The
need for protecting hyposmotic IF from freezing also applies to
other polar fishes; thus, we examined diverse species (eleven) from
both north- and south-polar (and subpolar) regions bearing all
other known types of AFs (species and AF information in Table 1).
Northern blots of RNA from fish bearing each type of AF hybrid-
ized with the corresponding AF cDNA (or gene) probe showed
pancreatic AF expression (Fig. 5 Upper) in addition to liver expres-
sion (Fig. 5 Upper and refs. 10–15). The pancreatic AFP mRNA of
type I, II, III, and IV AFP-bearing species corresponds in size to a
liver AFP mRNA (Fig. 5 A–D Upper), indicating expression of a
type III AFP mRNA in zoarcoid fishes (pouts and wolffish) (Fig.
5C), likely due to the tissue heterogeneity of the pancreatic isolates
and?or seasonal variation of synthesis in the northern hemisphere
species (23) (Atlantic ocean pout Macrozoarces americanus and
spotted wolffish Anarhichas minor sampled in late spring). Strong
pancreatic AFGP mRNA expression is seen in the three northern
kb, with predominant transcripts at ?2 kb for the saffron cod
Eleginus gracilis and ?3 kb and 9 kb for the high-latitude polar cod
Boreogadus saida and the icecod Arctogadus glacialis (Fig. 5E
Upper), reflecting AFGP multigene families and the large polypro-
tein structure of the transcribed genes (15). Regardless of hybrid-
ization intensity, all RNA samples yielded AF cDNAs upon RT-
the translated sequences of pancreatic and liver AFP cDNAs from
each species show high amino acid identities inclusive of the
conserved signal peptide (Fig. 7 A–E, which is published as sup-
hysteresis (TH, or AF activity) of intestinal, stomach, and pancreatic fluids of
Antarctic notothenioids and Antarctic eelpout. Figure is graphical represen-
tation of data in Table 2. Values of ambient McMurdo Sound seawater are
given at the top for reference. TH represented by gray box, and indicated
value is the difference between MP and FP in degrees Celsius.
Osmolarity, melting point (MP), freezing point (FP), and thermal
GI fluids. (A) Fluorescently labeled AFGPs purified from pancreatic fluid (PF),
serum of the same D. mawsoni specimen. (B) Coomassie blue-stained type III
and a small amount of the ?14-kDa isoform. Purified serum AFP isoform
counterparts (7-kDa RD2 and 14-kDa RD3) (24) were coelectrophoresed as
reference. Insulin (5.7 kDa) was used as a low molecular weight standard. The
?5.7-kDa peptide band in IF may be partly digested fragment of the 7-kDa or
14-kDa AFP isoform.
Cheng et al.
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porting information on the PNAS web site), indicating pancreatic
AFPs are secreted molecules. Likewise, a signal peptide sequence
precedes the AFGP encoded in the pancreatic AFGP cDNA from
the saffron cod (Fig. 7F); thus, gadid pancreatic AFGPs are also
In addition to verifying pancreatic expression at the transcrip-
fluids of the type III AFP-bearing Antarctic eelpout Lycodichthys
dearborni and determined the presence of stomach and intestinal
AFP on the basis of significant thermal hysteresis in the respective
fluid (Fig. 3). Furthermore, we isolated apparently intact 7-kDa
type III AFP from the IF similar to the serum isoform RD2 (24)
(Fig. 4B), supporting the entry of the secreted stomach and
pancreatic AFPs into the intestinal lumen. A ?5.7-kDa peptide is
also present in the IF, which may represent a partially digested
fragment of the 14-kDa (RD3) (24) or 7-kDa isoform.
of plasma proteins (8, 9), and that hepatic synthesis and secretion
is responsible for the high circulatory levels of AF protein in
AF-bearing polar and subpolar fishes (10–15), is based on docu-
mented evidence. It is therefore natural to assume that the same
applies to the bloodborne AFGPs of the Antarctic notothenioid
hepatic AFGP synthesis does not occur in the Antarctic notothe-
nioid fishes, the predominant teleost group endemic to the South-
ern Ocean (1). The absence of liver AFGP synthesis in the day-old
hatchling and 4-month-old G. acuticeps larva (Fig. 2) indicates that
no hepatic contribution to blood AFGPs occurs during the early
posthatch life stages, although blood AF in these AF-deficient
larvae steadily accumulates to adult levels during this time (from
this period of accrual to adult plasma AF concentrations, together
with the absence of hepatic AFGP expression in adults (Fig. 1A),
confirms that liver plays no role in AFGP biosynthesis throughout
the life of notothenioid fishes. Our Northern blot results indicating
the absence of hepatic AFGP synthesis in adults is corroborated by
expressed sequence tags (EST) sequencing projects of two adult
Antarctic notothenioid species included in our analysis (Fig. 1A),
D. mawsoni (L. Chen and C.-H.C.C., unpublished results) and
Harpagifer antarcticus (Melody Clark, British Antarctic Survey,
personal communication), which revealed no AFGP cDNA from
the liver. For D. mawsoni, none of the ?4,700 unique cDNAs
obtained from ?12,000 sequenced clones code for AFGP (L. Chen
and C.-H.C.C., unpublished results). Together with this study, we
have established a body of compelling evidence demonstrating the
AFGP-synthesis null condition of the Antarctic notothenioid liver.
Without hepatic AFGP synthesis, we come full circle to the
fundamental question of the source of the abundant AFGPs in the
blood, decades after the protein was discovered in the Antarctic
notothenioids (5). From the evidence in this study, we propose that
reabsorption of pancreatic- and stomach-derived intestinal AFGPs
is the source of circulatory AFGPs in Antarctic notothenioids,
through the following deduction. We have shown the exocrine
pancreas to be the major AFGP expression site from hatching to
adulthood by Northern blot and immunodetection (Figs. 1 and 2),
and Northern blot analysis reveals the anterior stomach as the only
additional site (Fig. 1). No other tissues we examined by Northern
blot analysis showed AFGP mRNA expression, including hemo-
poietic tissues (head kidney and spleen) and circulating blood cells
(data not shown) that could potentially contribute to bloodborne
PF, and IF (Fig. 3), and the isolation of similar complements of
AFGP isoforms from the PF and IF (Fig. 4A); both pancreatic and
gastric AFGPs expectedly reach the intestine through obvious
pancreas and anterior stomach only, and not from the components
of the lower GI tract, because pyloric ceca and intestine cleaned of
AFGP mRNA expression (Fig. 1 B and C). We observed that the
intestinal AFGPs remain largely undegraded as they transit the
acid and alkali by virtue of high sugar content (?60% by mass) and
the lack of cleavage sites for known digestive proteases in their
repetitive peptide backbone (Thr-Ala?Pro-Ala-)n (4, 22). The
nearly identical intestinal and serum AFGP profiles suggest that
serum AFGPs could be derived from intestinal AFGPs. Reuptake
mechanisms for intestinal AFGPs may exist because rectal uptake
of the protein macromolecule ferritin introduced into the GI tract
has been demonstrated for the Antarctic nototheniid Notothenia
neglecta (26), and a body of experimental evidence exists showing
intestinal absorption of macromolecular proteins for intracellular
Type II AFP, herring and smelt. (C) Type III AFP, zoarcoid fishes. Ma and Am are late spring specimens that have greatly reduced AFP synthesis. (D) Type IV AFP,
longhorn sculpin. (E) AFGP, northern hemisphere gadids. (Lower) Same blots hybridized with pancreatic trypsinogen cDNA to verify presence of exocrine
pancreas in pancreatic isolates. Faint trypsinogen hybridization in liver RNA (Pbr, Mo) is likely a result of pancreatic infiltration in liver (32). (Detailed species
information is in Table 1.)
Northern blot analysis of AF mRNA expression in pancreas and liver of nonnotothenioid AF-bearing fishes. (Upper) (A) Type I AFP, winter flounder. (B)
www.pnas.org?cgi?doi?10.1073?pnas.0603796103Cheng et al.
processing in teleost fishes (27). Collectively, the above reasoning
leads us to propose that pancreatic- and gastric-derived intestinal
AFGPs may be returned to the blood by intestinal and?or rectal
absorption. The secretion of pancreatic and gastric AFGPs, their
transit into the intestine, and subsequent intestinal?rectal absorp-
that would take considerable time, and some excretory loss of
intestinal AFGPs likely occurs. These factors conceivably contrib-
posthatching) observed in the nascent AF-deficient G. acuticeps
larvae (25). Once in the blood, the aglomerular kidneys, a derived
production (28, 29), serve to conserve AFGPs in the circulation.
Renal AFGP conservation and high chemical stability of the
AFGPs imply that, once sufficient plasma levels are achieved, only
maintenance levels of input of GI AFGPs to blood would be
necessary, which could be readily provided by the GI-to-blood
transport even if the process lacks efficiency. Inefficient GI-to-
blood transport would mean significant loss of intestinal AFGPs,
which seems energetically wasteful. Some fraction of intestinal
AFGPs may indeed be ‘‘recycled’’ via a blood-to-bile-to-intestine
pathway, because AF activity is found in the gall bladder bile of
notothenioids (results not shown; ref. 35). Given that the liver does
not synthesize AFGPs, bile AFGPs are likely derived from plasma
AFGP via paracellular and?or transhepatocyte blood-to-bile trans-
port in the liver, known to occur for plasma proteins in mammalian
appears to be a constitutive condition in notothenioids (regardless
of age and time of pancreatic tissue sampling), suggesting that
continuous resynthesis to replenish intestinal loss may be an oblig-
atory energetic cost for ensuring intestinal freezing avoidance.
Our discovery of the AFGP-synthesis active exocrine pancreas
fishes (6, 7, 16) is erroneous, and that the reported observations of
hepatic AFGP expression might be of pancreatic origin. The
exocrine pancreas in almost all adult teleost fishes, including
associated with surfaces of the abdominal organs, adipose tissue,
mesentery, blood vessels, and ducts, on and in the gall bladder wall,
and, in some fishes, it also infiltrates the liver along hepatic portal
blood vessels forming hepatopancreas islets (19, 20, 32). Thus, the
reported AFGP synthesis in primary cultures of isolated hepato-
cytes by radioactive labeling (7) and detection of liver AFGP
mRNA in Northern blot with an unusually large amount (50 ?g) of
1% mRNA abundance, as opposed to 5–15 ?g total RNA per
sample used in Northern blots in this study) could have resulted
the converging biliary ductules exiting the notothenioid liver (19).
in the blood behind non-AFGP plasma proteins in radioactive
tracer studies (6) could be reconciled by our proposal that the
source of plasma AFGPs is the secretion and reabsorption of
intestinal AFGPs, expectedly a slow process, and therefore not
synchronized with liver synthesis and secretion of other plasma
The historical focus of polar teleost freeze avoidance studies has
been the primary importance of preventing the blood from freez-
ing, ostensibly shaped by the conspicuously high plasma levels of
AFs in the adult fishes. Less obvious and far under-recognized is
that freezing prevention of the hyposmotic fluids of the alimentary
canal is equally crucial to survival. The endowment of AFs in the
stomach and intestinal fluids (particularly ample in the highly
hyposmotic IF) in unrelated Antarctic notothenioid and zoarcid
(eelpout) fishes (Figs. 3 and 4) and the functional convergence of
pancreatic AF expression in all AF-bearing fishes regardless of AF
type (Figs. 1 and 5) attest to the universal need for preventing the
hyposmotic GI fluids from freezing. This need inevitably arises
for osmoregulation (21, 33), which generates an avenue for the
notothenioids confined to chronically ice-laden freezing waters (3,
22) and for north-polar fishes when their habitats reach freezing
temperatures and become icy. Intake of ice-associated food in icy
GI fluids, which must be prevented to ensure organismal survival.
The presence of high levels of AFGPs in the IF of Antarctic
notothenioids has been recognized in earlier studies (34, 35), but
the source was unknown until the present study. For fishes that
possess other types of AF protein, presence of intestinal AF is
demonstrated for the type III AFP-bearing Antarctic eelpout L.
dearborni (Figs. 3 and 4B) in this study, and recently for the
AFGP-bearing Arctic ice cod A. glacialis (36), both of which show
pancreatic mRNA expression of their respective AF (see L. dear-
born and A. glacialis in Fig. 5). It follows that the intestinal AF
originates from pancreatic AF secretions through the direct pan-
creatic duct connection between the exocrine pancreas and intes-
tine in these species, as well as in fishes bearing type I, II, and IV
AFP (Fig. 5), which show pancreatic expression of AF mRNA
carrying a secretory signal sequence (Fig. 7). Furthermore, besides
Antarctic notothenioids, we found type III AFP mRNA expression
in the anterior stomach of L. dearborni (data not shown) and AF
activity in the stomach fluid (Fig. 3), and stomach expression of the
mRNA of the secreted type of AFP I by winter flounder has also
been observed (12), suggesting that this additional GI source of AF
may also be common among AF-bearing fishes.
The evolutionary confinement of Antarctic notothenioids to
persistently icy conditions in the Southern Ocean, where ice inges-
tion is recurrent and arresting ice growth in their hyposmotic GI
fluids a constant necessity (22), combined with the absence of liver
AFGP expression, suggests that natural selection acted first and
foremost on preventing GI freezing in these fishes. Consistent with
the selection for GI freeze avoidance is the evolution of the AFGP
gene from a pancreatic trypsinogen-like protease (TLP) gene (17,
18) and the exclusive role of GI components in AFGP expression
(this study). Experimentally, wild-caught notothenioid fishes ren-
dered free of associated ice by warming at 1°C acquire ice in their
IF much faster than in their blood after being returned to the
ambient icy waters of McMurdo Sound, Antarctica (22), under-
fluids over other body fluids. The eventual acquisition of ice in the
blood means continual protection of the hyposmotic vascular fluid
pancreas and anterior stomach and has not spread to the liver over
evolutionary time for more expeditious delivery of AFGPs into the
blood seems to defy reasonings of evolutionary parsimony. A
possibility resides in the expression of the evolutionarily related
AFGP and TLP genes being potentially still under common tran-
scriptional control, as indicated by the chimeric AFGP?TLP genes
that are transcriptionally active in the pancreas (18). Without
separate transcriptional control, concomitant expression of high
levels of a serine protease by the liver could lead to unfavorable
physiological consequences. Another aspect of Antarctic notothe-
nioid physiology regarding freeze avoidance, alluded to earlier, is
the AF-deficient (up to 3 months posthatching) but paradoxically
freeze-resistant condition of young larvae that apparently rely on
intact skin and under-developed gill structures as effective physical
barriers to ice entry during that time, instead of fully AFGP-
fortified blood (25). Collectively, these physiologies highlight a
general need for a greater awareness of evolutionary creativity in
This study establishes active pancreatic AFGP synthesis and the
AFGP-expression null state of the liver in Antarctic notothenioid
fishes, bringing a significant perspective to teleost freeze-avoidance
physiology. It also reveals that the long-held paradigm of hepatic-
Cheng et al.
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Our proposal of reabsorption of intestinal AFGP molecules as the
source of blood AF in Antarctic notothenioids is an experimentally
testable hypothesis that may further our knowledge of transport
physiology of macromolecules. Although freeze avoidance of the
blood remains crucial, our finding that divergent AF-bearing fish
of secreted AF regardless of AF type attests to freezing prevention
of the hyposmotic GI fluids as a vital component of teleost freeze
avoidance strategies. The universal pancreatic AF mRNA expres-
expectedly stimulate further investigations into the details of GI
freeze avoidance across other fish taxa and life stages.
Materials and Methods
Animals and Sampling. Fish were collected by traps or line through
sea ice or in open water, or by trawls from research vessels.
Blood was obtained from the caudal vein by using a needle and
syringe, and tissues were dissected, frozen in liquid nitrogen, and
stored at ?80°C. Intestinal fluid from unfed fish was drained
and collected from ligated intestines after they were blotted dry of
AF-bearing peritoneal fluid and removed from the abdomen.
Pancreatic fluid (up to ?400 ?l) from unfed D. mawsoni was
sampled with an insulin syringe from the pancreatic duct, which
distends into a visible small reservoir when filled with pancreatic
secretion. All animal handling was carried out in accordance with
institutionally approved protocols.
Northern Blot and RT-PCR Amplification. Total RNA was isolated
from tissues and 5–15 ?g per sample were used in Northern blot
analyses as described (15). Blots were stripped of hybridized probe
by using 0.1% SDS at 100°C before hybridization with a second
probe. Sequences of the primers used to RT-PCR amplify the
cDNA of each type of AF protein, elongation factor 1-? subunit,
and pancreatic trypsinogen are given in Table 3, which is
published as supporting information on the PNAS web site.
Amplified cDNA products were cloned into pGemTeasy (Pro-
mega) and sequenced with BigDye Terminator v.3 cycle se-
quencing (Applied Biosystems).
Osmolarity, Melting Point, and Freezing Point Determinations of Fish
pressure osmometer, and melting and freezing points were deter-
mined by using single crystal seed ice in a Clifton nanoliter
osmometer as described (25).
AFGPs were treated with trichloroacetic acid (5% final concen-
tration), and the acid-resistant AFGPs were purified from the
soluble fraction by dialysis and lyophilization. About 400 ?g of
purified AFGPs were fluorescently labeled with fluorescamine
polyacrylamide gel as described (15). Type III AFP from fluid
samples of Antarctic eelpouts were purified by G75 Sephadex
(Amersham Pharmacia) gel filtration column chromatography.
The AFP-containing column fractions were lyophilized and ?30
?g were electrophoresed on 15% SDS?polyacrylamide gel as
Immunodetection of AFGPs. Whole larvae or dissected tissues were
fixed immediately in cold 4% paraformaldehyde prepared in no-
tothenioid PBS (0.1 M sodium phosphate, pH 7.6, and adjusted to
450 mOsm with NaCl). Frozen sections (5–15 ?m) were pretreated
with PDB (notothenioid PBS containing 1% vol?vol DMSO and
1% wt?vol Ig-free BSA) for 20 min at 20°C, washed, and then
four times for 5 min with PDB and incubated for 1 h at room
temperature with an Alexa Fluor 546-labeled secondary goat
anti-rabbit IgG (Molecular Probes) diluted 1:1000 with PDB. After
0.2 ?g ml?1DAPI for 5 min at 20°C between the first and second
washes, the sections were mounted under a coverslip by using
FluoroGuard (Bio-Rad). Control sections were treated with pre-
immune serum instead of the primary antibody and were consis-
tently negative (results not shown).
We thank Clarabelle DeVries for assistance with the AFGP character-
ization and Vivian Ward with the immunodetection images. This work
was supported by U.S. National Science Foundation Office of Polar
Programs Grants OPP0002654 and OPP0231006 (to C.-H.C.C.). C.W.E.
acknowledges additional support from the University of Auckland
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